Within the scope of the present invention, a microcarrier or a microparticle refers to any type of particles, respectively to any type of carriers, microscopic in size, typically with the largest dimension being from 100 nm to 300 micrometers, preferably from 1 μm to 200 μm.
According to the present invention, the term microcarrier refers to a microparticle functionalized, or adapted to be functionalized, that is containing, or adapted to contain, one or more ligands or functional units bound to the surface of the microcarrier or impregnated in its bulk. A large spectrum of chemical and biological molecules may be attached as ligands to a microcarrier. A microcarrier can have multiple functions and/or ligands. As used herein, the term functional unit is meant to define any species that modifies, attaches to, appends from, coats or is covalently or non-covalently bound to the surface of said microcarrier or impregnated in its bulk. These functions include all functions that are routinely used in high-throughput screening technology and diagnostics.
Drug discovery or screening and DNA sequencing commonly involve performing assays on very large numbers of compounds or molecules. These assays typically include, for instance, screening chemical libraries for compounds of interest or particular target molecules, or testing for chemical and biological interactions of interest between molecules. Those assays often require carrying out thousands of individual chemical and/or biological reactions.
Numerous practical problems arise from the handling of such a large number of individual reactions. The most significant problem is probably the necessity to label and track each individual reaction.
One conventional method of tracking the identity of the reactions is achieved by physically separating each reaction in a microtiter plate (microarray). The use of microtiter plates, however, carries several disadvantages like, in particular, a physical limitation to the size of microtiter plates used, and thus to the number of different reactions that may be carried out on the plates.
In light of the limitations in the use of microarrays, they are nowadays advantageously replaced by functionalized encoded microparticles to perform chemical and/or biological assays. Each functionalized encoded microparticle is provided with a code that uniquely identifies the particular ligand(s) bound to its surface. The use of such functionalized encoded microparticles allows for random processing, which means that thousands of uniquely functionalized encoded microparticles may all be mixed and subjected to an assay simultaneously. Examples of functionalized encoded microparticles are described in the international patent application WO 00/63695 and are illustrated in FIG. 1.
The international patent application WO 2010/072011 describes an assay device having at least a microfluidic channel which serves as a reaction chamber in which a plurality of functionalized encoded microparticles or microcarriers 1 (FIG. 1) can be packed. The microfluidic channel is provided with stopping means acting as filters that allow a liquid solution containing chemical and/or biological reagents to flow through while blocking the microcarriers 1 inside. The geometrical height of said microfluidic channels and the dimensions of said microcarriers 1 are chosen so that said microcarriers 1 are typically arranged in a monolayer arrangement inside each microfluidic channels preventing said microcarriers 1 to overlap each other.
Those functionalized encoded microcarriers 1 that show a favorable reaction of interest between their attached ligand(s) and the chemical and/or biological reagents flowing through may then have their code read, thereby leading to the identity of the ligand that produced the favorable reaction.
The code may comprise a distinctive pattern of a plurality of traversing holes 2 and may also include an asymmetric orientation mark such as, for example, a L-shaped sign 3 (as shown in FIG. 1) or a triangle. This asymmetric orientation mark allows the distinction between the top surface 4 and the bottom surface 5 of the microcarrier 1.
The term microfluidic channel refers to a closed channel, i.e. an elongated passage for fluids, with a cross-section microscopic in size, i.e. with the smallest dimension of the cross-section being typically from about 1 to about 500 micrometers, preferably about 10 to about 200 micrometers. A microfluidic channel has a longitudinal direction, that is not necessarily a straight line, and that corresponds to the direction in which fluids are directed within the microfluidic channel, i.e. preferably essentially to the direction corresponding to the average speed vector of the fluid, assuming a laminar flow regime.
With the assay device described in WO 2010/072011, the detection of a reaction of interest can be based on continuous readout of the fluorescence intensity of each encoded microcarrier 1 present in a microfluidic channel, as depicted in FIG. 2. In other words, the presence of a target molecule in the assay will trigger a predetermined fluorescent signal. However, the predetermined fluorescent signal can be very difficult to detect due to the presence of strong fluorescent background.
It is known that coating the microcarriers with an optical layer increases the fluorescence emitted during the assay to a detectable level. For example, FIG. 2 shows a batch of coated microcarriers 1 obtained by the method described in the document WO 2011/044708, wherein an optical layer is deposited on the microcarriers 1.
However, the result of the biological assay illustrated in FIG. 2, shows different patterns of fluorescent signal emitted from the coated microcarriers 1. In particular, some microcarriers 1a emit a homogeneous and detectable fluorescent signal while other microcarriers 1b emit a partial or non-homogeneous fluorescent signal, which has most of time a shape of a crescent moon (hereafter referred to as “shadow effect”). Furthermore, some microcarriers do not emit any detectable fluorescence because they are exempt of optical layer on their surface.
Such defects render difficult the extraction of precise quantitative information during the analysis.
The absence or the partial deposition of the optical layer on some microcarriers 1b results from the process involved in the document WO 2011/044708. Indeed, this process cannot avoid the partial or full overlapping between several microcarriers 1 before and during the deposition of the optical layer. Such overlapping is shown in FIG. 3 where an area A of the top surface 4 of a microcarrier 1 will be coated by an optical layer, whereas an area B of the top surface 4 of said microcarrier 1, hidden by a another microcarrier 1′, will not be coated by said optical layer.
Furthermore, during the process described in WO 2011/044708, several microcarriers may flip over before coating and thus be coated on the wrong surface.
Moreover, it is impossible to separate the partly coated microcarriers 1b or the non-coated microcarriers from the well coated microcarriers 1a before performing the fluorescent assay. Indeed, the presence of an optical layer on a microcarrier is only distinguishable by a fluorescent signal emitted during the fluorescent assay.